This research program will the investigate use of pharmacologic ascorbate (high-dose, i.v. delivery of vitamin C) in the treatment of cancer. Pharmacological ascorbate (AscH-) takes advantage of the basic chemical properties of AscH- to use it as a drug; in fact because of its properties as a reducing agent, pharmacologic AscH- is a pro-drug for the delivery of extracellular H2O2 to tumor cells. In this use of AscH-, it must be given intravenously; plasma levels of 20 - 30 mM are achieved; healthy individuals have plasma ascorbate levels on the order of 50 ?M (0.05 mM). With pharmacological AscH- the goal is to achieve a transient level of ascorbate in plasma on the order of 300-500 times that of typical healthy nutritional levels. The half-life of AscH- in plasma at these high levels is H2.3 h. Thus for 12 - 24 h after treatment, levels of AscH- in plasma greatly exceed healthy nutritional levels. We propose to investigate the mechanism of action of pharmacological AscH- to learn: (1) what biochemical properties make cancer cells susceptible to pharmacological AscH-; and (2) why it is not toxic to normal tissue. Our goal is to unravel basic biochemical mechanisms so this therapy can be employed in a broad range of appropriately selected cancers. We hypothesize that the difference in susceptibility of cells to pharmacological AscH- is the ability o maintain their intracellular redox buffer (GSSG,2H+/2GSH) at a half-cell reduction potential (Ehc) compatible with life. The rationale for this hypothesis is that: (1) AscH- readily autoxidize producing a flux of H2O2 (in cell culture media and in vivo); (2) the high levels of extra cellular AscH- achieved by i.v. delivery (H300-500X nutritional levels) produce a high flux of H2O2; (3) the removal of this high flux of H2O2 by cells results in a great demand for intracellular reducing equivalents, i.e. glutathione (GSH) and NADPH; (4) this results in oxidation of the intracellular redox buffer, leading to quiescence or cell death, depending on the extent of oxidation. Cells that maintain an appropriately reduced intracellular redox buffer will be less susceptible to exposure to pharmacological AscH-; cells that cannot maintain their intracellular redox buffer will die. Because the status of the redox buffer is maintained by the pentose phosphate pathway (PPP), we further propose that an oxidatively challenged redox buffer will be synergistic with agents that also connect to the PPP, e.g. gemcitabine, 5-fluorouricil, and especially ionizing radiation. This research program supports translational efforts by addressing the fundamental question of why pharmacological ascorbate is non-toxic to organisms, i.e. people, yet cancer cells can be very susceptible. The results of this study will guide translational efforts in selectng appropriate adjuvants for therapy and cancers (patients) that may benefit from this approach to treatment.